How does the use of validated primers differ from the primers that were initially designed and how did the use of these change the results from what they would have been with the initial primers?
The primers that were initially designed did not successfully anneal to and amplify Rfc1, so we needed to use validated primers in order to move on with our project. The amplification of the target gene was necessary to analyze the effects of DNA damage, so using primers that could not anneal just came up with a random amplification. Thanks for your question!
DNA damage response and genome-editing go hand in hand, as editing processes such as CRISPR induce DNA damage in order to repair the cell with a specific and targeted revision (Nambiar et. al., 2022). So, since we know Rfc1 is involved in this pathway, this makes it a perfect gene of study in this regard! Thanks for your question!
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How does the use of validated primers differ from the primers that were initially designed and how did the use of these change the results from what they would have been with the initial primers?
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The primers that were initially designed did not successfully anneal to and amplify Rfc1, so we needed to use validated primers in order to move on with our project. The amplification of the target gene was necessary to analyze the effects of DNA damage, so using primers that could not anneal just came up with a random amplification. Thanks for your question!
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How exactly could you see Rfc1 being used in the future for gene editing specifically?
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DNA damage response and genome-editing go hand in hand, as editing processes such as CRISPR induce DNA damage in order to repair the cell with a specific and targeted revision (Nambiar et. al., 2022). So, since we know Rfc1 is involved in this pathway, this makes it a perfect gene of study in this regard! Thanks for your question!
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Why do you think there was contamination in lane 6?
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I think that there existed cDNA contamination in Lane 6, which could have been caused by improper PCR technique and/or lack of sterility.
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